Identification and Characterization of a Novel Thermostable and Salt-Tolerant ß-1,3 Xylanase from Flammeovirga pacifica Strain WPAGA1.

ß-1,3 xylanase is an important enzyme in the biorefinery process for some algae. The discovery and characterization of new ß-1,3 xylanase is a hot research topic. In this paper, a novel ß-1,3 xylanase (Xyl88) is revealed from the annotated genome of Flammeovirga pacifica strain WPAGA1. Bioinformatic analysis shows that Xyl88 belongs to the glycoside hydrolase 26 (GH26) with a suspected CBM (carbohydrate-binding module) sequence. The activity of rXyl88 is 75% of the highest enzyme activity (1.5 mol/L NaCl) in 3 mol/L NaCl buffer, which suggests good salt tolerance of rXy188. The optimum reaction temperature in the buffer without NaCl and with 1.5 mol/L NaCl is 45 °C and 55 °C, respectively. Notably, the catalytic efficiency of rXyl88 (kcat/Km) is approximately 20 higher than that of the thermophilic ß-1,3 xylanase that has the highest catalytic efficiency. Xyl88 in this study becomes the most efficient enzyme ever found, and it is also the first reported moderately thermophilic and salt-tolerant ß-1,3 xylanase. Results of molecular dynamics simulation further prove the excellent thermal stability of Xyl88. Moreover, according to the predicted 3D structure of the Xyl88, the surface of the enzyme is distributed with more negative charges, which is related to its salt tolerance, and significantly more hydrogen bonds and Van der Waals force between the intramolecular residues, which is related to its thermal stability.

Probing the Molecular Structure and Orientation of the Leaf Surface of Brassica oleracea L. by Polarization Modulation-Infrared Reflection-Absorption Spectroscopy.

The surface of most aerial plant organs is covered with the cuticle, a membrane consisting of a variety of organic compounds, including waxes, cutin (a polyester) and polysaccharides. The cuticle serves as the multifunctional interface between the plant and the environment, and plays a major role in protecting plants against various environmental stress factors. Characterization of the molecular arrangements in the intact cuticle is critical for the fundamental understanding of its physicochemical properties; however, this analysis remains technically challenging. Here, we describe the nondestructive characterization of the intact cuticle of Brassica oleracea L. leaves using polarization modulation-infrared (IR) reflection-absorption spectroscopy (PM-IRRAS). PM-IRRAS has a probing depth of less than several hundreds of nanometers, and reveals the crystalline structure of the wax covering the cuticle surface (epicuticular wax) and the nonhydrogen-bonding character of cutin. Combined analysis using attenuated total reflection-IR spectra suggested that hemicelluloses xylan and xyloglucan are present in the outer cuticle region close to the epicuticular wax, whereas pectins are dominant in the inner cuticle region (depth of ≤2 µm). PM-IRRAS can also determine the average orientation of the cuticular molecules, as indicated by the positive and negative spectral peaks. This unique advantage reveals the orientational order in the intact cuticle; the hydrocarbon chains of the epicuticular wax and cutin and the backbones of hemicelluloses are oriented perpendicular to the leaf surface. PM-IRRAS is a versatile, informative and easy-to-use technique for studying plant cuticles because it is nondestructive and does not require sample pretreatment and background measurements.

Identification and characterization of the first ß-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10.

In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a ß-d-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40°C. The enzyme was stable up to 40°C over a wide pH range (3.1-8.9). rSWU43A activity was enhanced by Fe2+ and Mn2+ and inhibited by various metals (Ag+, Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+), d-xylose, and l-arabinose. rSWU43A showed activity on p-nitrophenyl-ß-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside substrates, with specific activities of 0.09 and 0.06U/mg, respectively, but not on any xylosidic or arabinosidic polymers. rSWU43A efficiently degraded ß-1,3-xylooligosaccharides to produce xylose but showed little activity towards ß-1,4-xylobiose, with specific activities of 1.33 and 0.003U/mg, respectively. These results demonstrate that SWU43A is a ß-1,3-d-xylosidase (EC 3.2.1.72), which to date has only been described in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first ß-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool for the structural elucidation and production of xylose from ß-1,3-xylan in seaweed cell walls.

Expression of cold-adapted ß-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

Cold-adapted ß-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.

Biochemical characterization of a thermostable ß-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359.

The biochemical properties of a putative ß-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against ß-1,3-xylan and released ß-1,3-xylobiose and ß-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3-10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble ß-1,3-xylan K m value was 10.35 mg/ml and the k cat value was 588.24 s(-1). The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.

Mechanistic insights into the 1,3-xylanases: useful enzymes for manipulation of algal biomass.

Xylanases capable of degrading the crystalline microfibrils of 1,3-xylan that reinforce the cell walls of some red and siphonous green algae have not been well studied, yet they could prove to be of great utility in algaculture for the production of food and renewable chemical feedstocks. To gain a better mechanistic understanding of these enzymes, a suite of reagents was synthesized and evaluated as substrates and inhibitors of an endo-1,3-xylanase. With these reagents, a retaining mechanism was confirmed for the xylanase, its catalytic nucleophile identified, and the existence of -3 to +2 substrate-binding subsites demonstrated. Protein crystal X-ray diffraction methods provided a high resolution structure of a trapped covalent glycosyl-enzyme intermediate, indicating that the 1,3-xylanases likely utilize the (1)S(3) → (4)H(3) → (4)C(1) conformational itinerary to effect catalysis.

Expression, crystallization and preliminary X-ray diffraction studies of thermostable ß-1,3-xylanase from Thermotoga neapolitana strain DSM†4359.

Crystals of ß-1,3-xylanase (1,3-ß-D-xylan xylanohydrolase; EC 3.2.1.32) from Thermotoga neapolitana strain DSM 4359 with maximum dimensions of 0.2×0.1×0.02 mm were grown using the sitting-drop vapour-diffusion method at 293 K over 24 h. The crystals diffracted to a resolution of 1.82 Å, allowing structure determination. The crystals belonged to space group P2(1), with unit-cell parameters a=39.061, b=75.828, c=52.140 Å; each asymmetric unit cell contained a single molecule.

Inactive fluorescently labeled xylanase as a novel probe for microscopic analysis of arabinoxylan containing cereal cell walls.

A new technique to visualize cereal cell walls by fluorescence microscopy was developed. The novel staining technique is based on an inactive fluorescently labeled xylanase binding to arabinoxylan (AX), an important polysaccharide in grain cell walls in terms of the technological and physiological functionalities of grain. The xylanase probe could stain AX in the seed coat, nucellar epidermis, aleurone layer, and starchy endosperm, but not the highly substituted AX of the pericarp layer. The advantage of this new staining technique over the existing immunolabeling techniques is that the staining procedure is clearly faster and less laborious, and uses a smaller probe that can easily be produced by marking a well characterized enzyme with a fluorescent label. In the future, the here proposed technology can be used to develop probes having specificity also for cell wall components other than AX and thus to study plant cell walls further through fluorescence microscopy.

Catalytic performance of corn stover hydrolysis by a new isolate Penicillium sp. ECU0913 producing both cellulase and xylanase.

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.

Fusion of the OsmC domain from esterase EstO confers thermolability to the cold-active xylanase Xyn8 from Pseudoalteromonas arctica.

The OsmC-region (osmotically induced protein family) of the two-domain esterase EstO from the psychrotolerant bacterium Pseudoalteromonas arctica has been shown to increase thermolability. In an attempt to test if these properties can be conferred to another enzyme, we genetically fused osmC to the 3'-region of the family 8 xylanase encoding gene xyn8 from P. arctica. The chimeric open reading frame xyn8-OsmC was cloned and the chimeric protein was purified after heterologous expression in Escherichia coli. Xyn8 and Xyn8-OsmC showed cold-adapted properties (more than 60% activity at 0°C) using birchwood xylan as the preferred substrate. Maximal catalytic activity is slightly shifted from 15°C (Xyn8) to 20°C for Xyn8-OsmC. Thermostability of Xyn8-OsmC is significantly changed in comparison to wild-type Xyn8. The OsmC-fusion variant showed an apparent decrease in thermostability between 40 and 45°C, while both proteins are highly instable at 50°C.

Co-immobilization mechanism of cellulase and xylanase on a reversibly soluble polymer.

Cellulase and xylanase from Trichoderma reesei were immobilized simultaneously on Eudragit L-100, a reversibly soluble polymer. The effects of polymer concentration and polymer precipitation pH on enzyme activity recovery were investigated at an enzyme complex concentration of 1%. The immobilization mechanism of cellulase and xylanase on the polymer was discussed. An activity recovery of 75% and 59% was obtained for the cellulase and the xylanase, respectively, under the condition of a polymer concentration at 2% and a polymer precipitation pH at 4.0. Most zymoproteins might be connected to the polymer by electrostatic attraction in a medium of pH 4.8. In addition, the covalent coupling between the zymoproteins and the polymer was demonstrated by the infrared spectrograms. It was suggested that dehydration-condensation reaction occurred between the zymoproteins and the polymer during the immobilization.

Characterization and application of carbohydrate-binding modules of beta-1,3-xylanase XYL4.

beta-1,3-Xylanase from Vibrio sp. strain AX-4 (XYL4) is a modular enzyme composed of an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules (CBMs) belonging to family 31 in the C-terminal region. To investigate the functions of these three modules, five deletion mutants lacking individual modules were constructed. The binding assay of these mutants showed that a repeating unit of the CBM was a non-catalytic beta-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble beta-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble beta-1,3-xylan, but not to beta-1,4-xylan, Avicel, beta-1,4-mannan, curdlan, chitin or soluble glycol-beta-1,3-xylan. Both the enzyme and the binding activities for insoluble beta-1,3-xylan but not soluble glycol-beta-1,3-xylan were enhanced by NaCl in a concentration-dependent manner, indicating that the CBMs of XYL4 bound to beta-1,3-xylan through hydrophobic interaction. This property of the CBMs was successfully applied to the purification of a recombinant XYL4 from the cell extracts of Escherichia coli transformed with the xyl4 gene and the detection of beta-1,3-xylan-binding proteins including beta-1,3-xylanase from the extract of a turban shell, Turbo cornutus.

Factors affecting ferulic acid release from Brewer's spent grain by Fusarium oxysporum enzymatic system.

In this study, the factors affecting ferulic acid (FA) release from Brewer's spent grain (BSG), by the crude enzyme extract of Fusarium oxysporum were investigated. In order to evaluate the importance of the multienzyme preparation on FA release, the synergistic action of feruloyl esterase (FAE, FoFaeC-12213) and xylanase (Trichoderma longibrachiatum M3) monoenzymes was studied. More than double amount of FA release (1 mg g(-1) dry BSG) was observed during hydrolytic reactions by the crude enzyme extract compared to hydrolysis by the monoenzymes (0.37 mg g(-1) dry BSG). The protease content of the crude extract and the inhibitory effect of FA as an end-product were also evaluated concerning their effect on FA release. The protease treatment prior to hydrolysis by monoenzymes enhanced FA release about 100%, while, for the first time in literature, FA in solution found to have a significant inhibitory effect on FAE activity and on total FA release.

Purification and characterization of a thermostable xylanase from the brown-rot fungus Laetiporus sulphureus.

A thermostable extracellular xylanase was purified and characterized from brown-rot basidiomycete Laetiporus sulphureus, cultivated on biologically pretreated Pinus densiflora biomass. After three consecutive purification steps using DEAE, Mono Q, and Superdex 75 columns, the xylanase specific activity was found to be 72.4 U/mg, nine fold higher than that of the crude culture solution, purity was 96%, and the molecular mass determined to be 69.3 kDa. The optimal pH and temperature for xylanase activity were 3.0 and 80 degrees C, respectively. Although activity of xylanase was highest at 80 degrees C, it showed highest thermostability at 60 degrees C, retaining approximately 97% of its relative activity following incubation for 4 h. In the presence of 5 mM solution of CaCl2, the relative xylanase activity increased by 35.9%; however, it decreased significantly in the presence of 10 mM solution of Cu2+. Among the xylan-based substrates tested, purified L. sulphureus xylanase showed the highest activity on beechwood xylan. Thin-layer chromatography (TLC) experiments revealed that purified L. sulphureus xylanase is an endoxylanase that hydrolyzes xylotriose, xylotetraose, and xylopentaose but not xylobiose.

Characterization of a novel polyphenol-specific oligoxyloside transfer reaction by a family 11 xylanase from Bacillus sp. KT12.

A culture filtrate of Bacillus sp. KT12 was used to prepare polyphenyl beta-oligoxylosides from xylan and polyphenols in a one-step reaction. One oligoxyloside transfer enzyme was purified from multiple xylanolytic enzymes in the culture filtrate. N-terminal amino acid sequence determination classified the enzyme as a glycosyl hydrolase family 11 (endo-xylanase). The xylanolytic enzyme activities could be markedly altered; its hydrolytic activity was almost entirely inhibited at acidic pH, whereas near constant transxylosylation activity was observed at pH 4-11. Further, metal ions activated transxylosylation and almost completely inhibited hydrolysis. The enzyme specifically induced a beta-xylosyl transfer reaction to acceptor molecules, such as divalent and trivalent phenolic hydroxyl groups, and displayed no activity toward alcoholic compounds. The Bacillus sp. KT12 xylanolytic enzyme was a suitable enzyme for the synthesis of polyphenyl beta-oligoxylosides.

The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.

BACKGROUND: Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature. METHODOLOGY: We investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX. CONCLUSIONS: A single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering.

Shifting pH optimum of Bacillus circulans xylanase based on molecular modeling.

Although hydrolases are used in several industrial processes, its industrial applications have some limitations in specific cases since some industrial processes are carried out at pH value which is different from optimum pH of the enzyme. Alkaline side optimum pH of hydrolases is always desirable, and it is proved difficult to achieve that by mutation only. Hence, molecular modeling was applied to select the promising mutants. The changes in electrostatic potential, which was calculated using Delphi, were compared to the changes in pH optimum of four hydolases and their mutants. The results showed that the change in electrostatic potential can be used as an indicator for selecting relevant candidates of mutation. Bacillus circulans xylanase was selected as a model system, and the promising mutants were picked up by the molecular modeling. Q167M and R73V, had a higher pH optimum than the wild type, while K175Q had a similar pH-activity profile of the wild type.

Cloning of a novel gene encoding beta-1,3-xylosidase from a marine bacterium, Vibrio sp. strain XY-214, and characterization of the gene product.

The beta-1,3-xylosidase gene (xloA) of Vibrio sp. strain XY-214 was cloned and expressed in Escherichia coli. The xloA gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular weight of 60,835. The recombinant beta-1,3-xylosidase hydrolyzed beta-1,3-xylooligosaccharides to D-xylose as a final product.

Kinetic and thermodynamic study of a chemically modified highly active xylanase from Scopulariopsis sp: existence of an essential amino group.

The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer from Scopulariopsis sp. were chemically modified, resulting in charge neutralization and reversal. Modification of the second amino group was accompanied by the complete loss of enzyme activity in both the absence and presence of xylose. Multiple alignments of family 10 and 11 xylanases revealed that there is a pair of fully conserved Lys residues only in family 10 members. Xylanase structures from family 10 members showed that one of the conserved Lys residues is found near the active-site cleft that makes an H-bond with the substrate. The LAX and HAX isoenzymes in which one amino and three to four carboxyl groups were modified were subjected to kinetic and thermodynamic characterization. There were no differences in pH optima between the native and modified HAX, but there was a broadening of pH optimum toward the alkaline range for charge-neutralized LAX and a double pH optimum for charge-reversed LAX. TheV max/K m of both modified LAX and HAX decreased relative to the native species. The thermodynamics of xylan hydrolysis showed that the decrease in the catalytic activity of modified LAX enzymes was entropically driven. When compared with native enzyme, the thermostabilities of modified LAX enzymes increased in the presence and decreased in the absence of substrate. The thermodynamics of kinetic stability for modified LAX enzymes revealed that this increase in thermolability was owing to the decrease in DeltaH# with a concomitant increase in DeltaS# compared with native LAX. The thermostabilities of all the modified HAX species decreased except that of charge-neutralized HAX, whose half-life significantly increased in 50% (v/v) aqueous dioxan. These results suggest that the altered properties of the modified enzymes were a result of the conformational changes brought about by chemical modification.

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan.

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.